In the pathways for taste and smell, there is a transduction of an external chemical signal into an intracellular one in the receptor cell, usually a neuron or modified neuron. Chemical cues bind to the surface of the receptor cell and this interaction somehow results in increased neuronal activity or release of neurotransmitter. The transduction process between binding and electrical change appears to involve internal second messengers, such as cyclic nucleotides, calcium or lipid metabolites. Indeed, there are human disease states in which the transduction processes are defective at the level of G proteins, those proteins that shuttle information between some receptors and the enzymes or ion channels that produce the second messengers. Paramecia are single celled animals that have been likened to swimming neurons and they serve as examples of chemoreceptor cells. They change their swimming behavior to accumulate in attractant stimuli. The groundwork is laid to use Paramecium to explore its chemosensory signal transduction pathways: the coupling of its chemoreceptors, the roles of internal calcium levels and pH, and the calcium transport pump that is implicated in producing the hyperpolarizing membrane conductance of Paramecium chemoreception. In Paramecium, some attractant stimuli such as cAMP and folate bind to cell surface receptors and initiate a signal transduction pathway that leads to a change in membrane potential, a hyperpolarization generated not by a channel but most likely by a plasma membrane calcium pump. Other attractants appear to affect membrane potential through internal pH. To explore the receptor/pump coupling and the roles of internal calcium and pH in chemoreception, we propose a series of experiments that will utilize molecular genetic, electrophysiological, ion-sensitive dye, and motion analysis techniques. Specifically, we will uncouple the receptor and pump using antisense motion oligonucleotide techniques to down regulate the receptor, calmodulin, or the pump. The receptor will be immunoprecipitated to ascertain which proteins physically associate with it. We will transform cells with the Ca-ATPase gene and determine the consequences for stimulus induced conductances and chemosensory behavior. In order to begin to understand the second sensory transduction pathway, we will directly measure and manipulate internal pH and assess the effects on membrane electrical properties and behavior. The dissection of the chemosensory signal transduction pathways in Paramecium will lead to a better understanding of sensory transduction in general and perhaps to mechanisms of signalling that had not previously been described.